(1) Field of the Invention
This invention generally relates to the field of virology and, more particularly, to inducible viral replicons and to their use as expression vectors for making recombinant proteins and for detecting other viruses.
(2) Background of the Invention
Replicon RNAs are self-replicating RNA molecules that contain the genetic information needed for virus replication but frequently lack one or more of the genes encoding the structural proteins needed for virus assembly. There have been numerous reports of using replicons derived from alphaviruses such as Sindbis virus and Semliki Forest virus (SFV) as vectors for gene expression. See e.g., Bredenbeek et al., Semin. Virol. 3:297-310; 1992; Berglund et al., Bio/Technology 11:916-920, 1993; and Schlesinger, S., Mol. Biotechnol. 3:155-165, 1995. Alphaviruses are particularly suited for such use because these positive-strand RNA viruses have a broad host range, replicate exclusively in the cytoplasm of infected host cells, and their genomic RNA alone is sufficient to initiate replication and productive infection (Polo et al., Nature Biotechnol. 16:517-518, 1998). The original studies involved preparing a cDNA of an RNA replicon encoding a heterologous protein instead of the structural proteins, in which transcription of the cDNA was under the control of the bacteriophage SP6 promoter, transcribing the cDNA in vitro and transfecting the RNA transcripts into susceptible cultured cells (Xiong et al., Science 243:1188-1191, 1989).
More recently, several reports have described transcribing replicon cDNAs from promoters recognized by RNA polymerase II after transfection of the cDNA into cells (Dubensky et al., J. Virol. 70:508-519, 1996; Hariharan et al., J. Virol. 72:950-958, 1998; Herweijer et al., Human Gene Therapy 6:1161-1167, 1995; Berglund et la., Nature Biotechnol 16:562-565, 1998; and Polo et al., supra). However, because these constructs are constitutively expressed in the host cells, this approach could not be used to stably transform cells with a replicon cDNA derived from a cytopathic alphavirus which rapidly shuts down host protein synthesis and kills the host cell. Stable transformation of cells has been accomplished using a replication-defective Sindbis virus cDNA (Olivo et al., Virol. 198:381-384, 1994; U.S. Pat. No. 5,591,579). However, this strategy requires infection with Sindbis virus or transfection with Sindbis replicon RNA to obtain expression of the heterologous protein encoded by the Sindbis virus defective genome.
U.S. Pat. No. 5,789,245 to Dubensky et al., states that a stably transformed host cell line can be achieved by placing a replicon cDNA under the control of a transcriptionally inactive, but inducible, promoter. The only examples of such inducible replicon cDNAs that were specifically described in this reference are replicon cDNAs containing viral or cellular promoters stated to be active only in terminally differentiated cells. The reference states that such constructs can be used to transform undifferentiated cells with induction of the replicon requiring addition of a cell differentiation agent such as retinoic acid. However, the only such construct actually made contained the U3 region of the long terminal repeat (LTR) from Moloney murine leukemia virus (Mo-MLV) and when this luciferase-encoding replicon cDNA was transfected into undifferentiated F9 cells, luciferase expression above background (i.e., mock infected cells) was detected (FIG. 14), suggesting that at least some basal transcription from the Mo-MLV LTR occurred.
Most inducible systems can tolerate a certain amount of basal transcription either because it is several orders of magnitude lower than induced levels or because the level of basal transcription does not result in detectable levels of the gene product. If, however, the transcribed product is a replicon derived from a cytopathic RNA virus, a single molecule of functional replicon RNA that finds its way to the cytoplasm will initiate an autocatalytic cycle of RNA replication and transcription which will result in the inhibition of host protein synthesis and cell death. Thus, generation of stable cell lines containing an inducible promoter operably linked to a replicon cDNA derived from a cytopathic RNA virus requires that the promoter be completely silent in the absence of induction. However, in addition to the apparent basal transcription of the replicon in the '245 patent, this reference did not describe any experiments or show any data to establish that stably transformed cells could actually be achieved with the Mo-MLV-replicon cDNA or with constructs containing any of the listed inducible promoters. This reference also did not disclose or suggest using a silent promoter inducible by a DNA virus to launch an RNA replicon from a transfected cDNA and, furthermore, did not disclose or suggest packaging the launched RNA replicon to produce RNA virus particles that infect surrounding cells and thereby achieve intercellular amplification of the amount of heterologous protein synthesized whose expression was originally induced by the DNA-virus. Moreover, the '245 patent did not teach or suggest the use of any of its constructs for the detection of DNA viruses.
U.S. Pat. No. 5,418,132 to Olivo disclosed a method for detecting an infectious herpes virus which employs a cell line stably transformed with a chimeric gene having a reporter gene under the control of a promoter from the herpes virus that is induced by transactivating substances produced by the infecting herpes virus. The reference states that promoters from beta, or early herpes virus genes are preferred because of their ability to be transactivated. However, this reference did not disclose or suggest using beta-gene promoters from herpes virus to induce transcription of a replicon cDNA encoding a heterologous protein upon response to herpes virus infection nor did it teach using a second virus to achieve intercellular amplification of the signal induced by the herpes virus. Moreover, the skilled artisan could not have predicted from the data presented in the '132 patent whether beta-gene promoters from herpes virus would have the requisite degree of regulatory stringency when operably linked to a replicon cDNA to allow stable transformation of cells with replicon cDNAs derived from a cytopathic RNA virus.
It would be desirable therefore, to provide a means for launching a replicon RNA in a cell stably transformed with a cDNA of the replicon where the replicon is not transcribed in a cell in the absence of an inducing agent. It would also be desirable if such a stably transformed cell could be used for detecting the presence of infectious DNA viruses with the sensitivity and specificity necessary for a diagnostic assay.